RNA-Seq of homo sapiens: SMA astrocytes
RNAconcentrationsandRINvalueswereobtainedusingtheRNAScreenTapekitandtheTapestationsystem(Agilent);allsampleshadaRINscore>9withclear18Sand28Speaks.ERCCSpike-InControls(Invitrogen)wereaddedtoRNAsamples(inputRNAwas100ngpersample)priortoprocessingsamplewiththeNEBNextPoly(A)mRNAMagneticIsolationModule(NEB#E7490),NEBNextUltraIIRNALibraryPrepKitforIllumina(NEB#E7770)accordingtomanufacturersprotocol.NEBNextMultipleOligosforIlluminaSet1(NEB#E7335),2(NEB#E7500)and3(NEB#E7710)wereusedtocreateuniqueidentifiersforeachcDNAlibrarysample.QualityofcDNAlibrarieswereassessedusingtheDNA100ChipkitandTapestationsystem(Agilent).SingleandpooledlibrarieswasassessedusingtheNEBNextLibraryQuantkitforIllumina(NEB#E7630),Bio-RadCFX384realtimethermocyclerandcalculatedusingtheNEBqPCRwebtool(https://nebiocalculator.neb.com/qPCR).Onthedayofsequencing,samplesweredenaturedanddilutedfollowingtheIlluminaNextSeqsystemDenatureandDiluteLibrariesGuideusingthebead-basednormalizationmethodwithPhiXlibrarycontrol.Samplesweresequenced(20-30millionreaddepth,38bppairedendreads)ontheIlluminaNextSeqsystem.FASTQfileswereprocessedusingExpressioncount(STAR)pipelineinBasepair